Three RNA samples per differentiation stage were reverse transcribed according to the manufacturer’s instructions using PrimeScrip RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara) and miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, Beijing, China), respectively. The CFX Connect Real-Time PCR Detection System was used to perform qRT-PCR using TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara) and miRNA Plus miRNA qPCR Kit (TIANGEN), respectively. Additional file 13 contains a list of the primers used in qRT-PCR. Meanwhile, β–actin and U6 was used as the normalization controls, respectively. The 2-ΔΔCT method [76 (link)] was used to calculate the relative expression levels.
Primescrip rt reagent kit with gdna eraser perfect real time
Manufactured by Takara Bio
Sourced in China
The PrimeScrip RT reagent Kit with gDNA Eraser (Perfect Real Time) is a molecular biology tool designed for reverse transcription and real-time PCR. The kit includes reagents for efficient cDNA synthesis and the removal of genomic DNA contamination.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Mircute plus mirna first strand cdna kit, by Tiangen Biotech (1 mentions)
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (1 mentions)
Cfx connect real time pcr detection system, by Takara Bio (1 mentions)
Abi stratagene mx3000p instrument, by Agilent Technologies (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Takara minibest plant rna extraction kit, by Takara Bio (1 mentions)
Abi viia 7 dx system, by Thermo Fisher Scientific (1 mentions)
Nanodrop system, by Implen (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
3 protocols using primescrip rt reagent kit with gdna eraser perfect real time
1
Quantitative RNA Expression Analysis
2
Skin RNA Extraction and qRT-PCR Analysis
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The total RNA was extracted from the skin of the KO and control groups using TRIzol reagent (Thermo Fisher Scientific, ShangHai). PrimeScrip RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Biomedical Technology, Beijing) was used to obtain the cDNA. Real-time PCR was performed on ABI Stratagene Mx3000P instrument (Agilent Technologies, Santa Clara, CA, USA) using a TB Green Premix Ex Taq II (Takara Biomedical Technology, Beijing). The primer sequences used in this experiment are shown in Supplementary Table S3 . The expression levels were analyzed using the 2ΔΔCt method, and normalized against GAPDH. Each sample was run in triplicate.
3
Validating Dietary Fiber DEGs via qRT-PCR
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Candidate differentially expressed genes (DEGs) involved in dietary fiber metabolism were selected for validation by real time quantitative PCR (qRT-PCR). Total RNA was extracted through a TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, Inc., Dalian, China). RNA quality was evaluated by agarose gel electrophoresis and the NanoDrop system (Implen, Los Angeles, CA, USA). cDNA was synthesized with the PrimeScrip RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Inc., Dalian, China). The primer sequences used for qRT-PCR are listed in Table S1. qRT-PCR was performed on an ABI ViiA 7 DX system (Applied Biosystems) using SYBR Premix Ex Taq II (TaKaRa) with the ubiquitin gene as an endogenous control. Data analysis was performed using the 2 -ΔΔCT method. Values for mean expression and standard deviation (SD) were calculated from the results of three independent experiments.
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